Process for Making Mineralized Mycelium Scaffolding and Product Made Thereby

ABSTRACT

The process of making a mineralized mycelium scaffolding requires obtaining a scaffold of fungal biopolymer having a network of interconnected mycelia cells, functionalizing the biopolymer to create precursor sites and thereafter mineralizing the scaffold with one of silicates, apatites and carbonates. The mineralized mycelium scaffolding may be used for medical applications in place of mineralized collagen membranes and collagen/hydroxyapatite composite scaffolds.

This application claims the benefit of Provisional Patent Application 62/667,793 filed May 7, 2018.

This invention relates to a process for making a mineralized mycelium scaffolding and product made thereby.

As is known, mineralized collagen membranes are known to be useful for various medical applications. For example, U.S. Pat. No. 6,300,315 describes a mineralized collagen membrane consisting essentially of a collagen component and a calcium phosphate minerals component and describes various processes for making the . mineralized collagen membrane. Also, U.S. Pat. No. 7,514,248 describes various processes for making a composite comprising an organic fluid-swellable matrix, such as collagen, and a mineral phase, such as calcium carbonate or phosphate mineral phase, for use as a biomimetic of bone.

U.S. Pat. No. 5,532,217 describes a process for the mineralization of collagen fibers for use in bone replacement therapy and to induce repairs in bony defects.

U.S. Pat. No. 7,179,356 describes a process for preparing surfactant-polycrystalline inorganic nanostructured materials having designed microscopic patterns.

As is also known, bone implants may be provided with a coating of calcium phosphate for improved biocompatibility and bone adhesion, such as described in US 2012/0270031. Likewise, it has been known from US 2007/0196509 to use nanoscale hydroxyapatite particles for use in medical application and from US 2010/0158976 to make a collagen/hydroxyapatite composite scaffold that forms a base upon which tissue, such as cartilage, may be engineered.

Typically, the various processes for producing products as described above have required complex and time consuming steps.

Accordingly, it is an object of the invention to provide a relatively simple process for producing a mineralized biocompatible material.

It is another object of the invention to provide a unique biomedical material.

As described in published US Patent Application US 2015/0033620 (A), the environmental conditions for producing the mycological biopolymer product described therein, i.e. a high carbon dioxide (CO₂) content (5% to 7% by volume) and an elevated temperature (from 85° F. to 95° F.), prevent full differentiation of the fungus into a mushroom. There are no stipe, cap, or spores produced.

As described, a mycological biopolymer product consisting entirely of fungal mycelium is made by inoculating a nutritive substrate with a selected fungus in a sealed environment except for a void space, which space is subsequently filled with a network of undifferentiated fungal mycelium. The biopolymer product grows into the void space of the tool, filling the space with an undifferentiated mycelium chitin-polymer, which is subsequently extracted from the substrate and dried.

As further described in published US Patent Application US 2015/0033620 (A) the use of a lid may be used to control the localized environmental conditions influencing the growth of the mycological biopolymer.

As described in U.S. patent application Ser. No. 16/690,525, another method of growing a biopolymer material employs incubation of a growth media comprised of nutritive substrate and a fungus in containers that are placed in a closed incubation chamber with air flows passed over each container while the chamber is maintained with a predetermined environment of humidity, temperature, carbon dioxide and oxygen. The growth media in each container is incubated for a period of time sufficient for the fungus to digest the nutritive substrate and produce a mycelium biopolymer consisting entirely of fungal mycelium in each container.

Briefly, the invention provides a process wherein a scaffold of fungal biopolymer having a network of interconnected mycelia cells is initially obtained, for example, a scaffold of a mycological biopolymer made in accordance with published US Patent Application US 2015/0033620 and/or U.S. patent application Ser. No. 16/690,525.

Next, in accordance with the process, the scaffold of fungal mycelium is mineralized to apply one of a hydroxyapatite coating, calcite coating and silicate coating on the cells within the network.

The process comprises the steps of:

-   -   1. Preparing the fungal biopolymer as necessary     -   2. Using fungal biopolymer, functionalizing the fungal polymer         to create the necessary precursor site as necessary.     -   3. Mineralizing the fungal biopolymer, choosing a pathway with         respect to the specific mineral formation desired, including,         but not limited to, silicates, apatites, and carbonates.     -   4. If necessary, rinse and dry material, using methods suited         toward need.

EXAMPLES 1A Hydroxyapatite Mineralization of Mycelial Scaffold via a Solution-Based Reaction

When low-energy hydroxyapatite [Ca₅(PO₄)₃(OH)] mineralization is desired _(upon) a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents (cleaning while preserving structure). This will serve as the raw scaffold, upon which mineralization will occur.

The cleaned scaffold is then phosphorylated, functionalizing the chitin/chitosan backbone, replacing the —OH moiety with a phosphate group, thereby increasing the scaffold's affinity for cations to prepare the scaffold for mineralization.

The scaffold should be thoroughly rinsed of any interfering residue and is then attached to a nonreactive clip to be freely suspended in and/or imbibed with in a saturated calcium hydroxide solution for 1-30 days, depending on desired degree of calcification, purity of starting material, degree of phosphorylation, or the like. In this step, calcium penetrates the scaffold which creates the calcium phosphate precursor sites necessary for hydroxyapatite formation.

After rinsing off residual calcium hydroxide solution, the scaffold is suspended in a 36.5° C. solution with ion concentrations 1.5 times that of the human body (1×=Na⁺—142.0 mM; K⁺—5.0mM; Mg²⁺—1.5 mM; Ca²⁺—2.5 mM; Cl⁻—148.8 mM; HCO₃ ⁻—4.2 mM; HPO₄ ²⁻—1.0 mM; trishydroxymethyl aminomethane—50 mM). The salts used to create this solution are: NaCl, NaHCO₃, KCl, K₂HPO₄.3H₂O, MgCl₂.6H₂O, CaCl, trishydroxymethyl aminomethane.

Once prepared, the solution is buffered to pH 7.25 with concentrated HCl. To ensure process consistency, the ion concentration should be monitored and adjusted regularly either with a chemostat or manual inspection. This step requires upwards of 30 days of active soaking of the scaffold (with or without initial imbibing) to precipitate a hydroxyapatite coating of the desired thickness on the cells of the mycelial mass of the scaffold.

The mineralized scaffold should then be dried, completing this mineralization process. The resultant scaffold of fungal biopolymer has a network of interconnected mycelia cells and a hydroxyapatite coating on the cells within the network.

1B Hydroxyapatite Mineralization of Mycelial Scaffold via a Solid-State Reaction

When hydroxyapatite [Ca₅(PO₄)₃(OH)] mineralization is “quickly” desired _(upon a) bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

A hydroxyapatite slurry is prepared by milling calcium carbonate and dicalcium phosphate in deionized water (DI) until most agglomerated particles are destroyed.

This slurry is then dried until most residual moisture has been removed to form a desiccated powder.

The desiccated powder is then calcinated at 900° C. for 1 hour at a heating rate of 5° C./min. This reaction creates a hydroxyapatite powder.

The next step involves creating a ceramic slurry with the powder, DI water, a plasticizer (including, but not limited to, polyethylene glycol, glycerin, sorbitol, alkyl citrates, or acetylated monoglycerides), a binder (including, but not limited to: polyvinyl alcohol, lecithin, soy lecithin, or sodium stearoyl lactylate), and dispersant (including, but not limited to, polycarboxylate ether based superplasticizers, or Dispex polyacrylate dispersant).

The ceramic slurry should be created according to the following percentages: hydroxyapatite—54 wt %, DI water—33.8 wt %, plasticizer—6.2 wt %, binder—4.4 wt %, and dispersant—1.6 wt %. This is milled for 24 hours to destroy agglomerated particles.

The prepared scaffold is then imbibed with the milled ceramic slurry via vacuum infusion and lyophilized to remove moisture. The dry slurry/scaffold matrix is then sintered in a 1300° C. in a furnace for four hours at a heating rate of 5° C./min., creating a ceramic in the form of the original fungal scaffold.

The resultant ceramic consists of a scaffold of fungal biopolymer with a network of interconnected mycelia cells and a hydroxyapatite coating on at least some of the cells within the network.

1C Hydroxyapatite Mineralization of Fungal Biopolymer Scaffold via a Solution-Based Reaction

When low-energy hydroxyapatite [Ca₅(PO₄)₃(OH)] mineralization is desired _(upon) a scaffold consisting of aligned bundles of fungal biopolymer assembled into a desired microstructure (e.g., helicodical to increase compressive strength by hindering crack propagation), one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur. From here, the procedure from 1A, beginning with functionalizing the chitin/chitosan backbone, followed through completion.

The scaffolds mineralized with hydroxyapatite may be of a size and shape to be put to use as a biomedical material, for example, the scaffold may be of a flat panel shape with a thickness of 2.5 cm.

The mineralized scaffolds can be as small as 1 mm×1 mm×1 mm, and the largest piece that has been created is 15 cm×5 cm×2.5 cm.

2A Calcite Mineralization of Mycelial Scaffold via a Solution-Based Reaction

When a low-energy calcite (CaCO₃) mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

A supersaturated solution is prepared from filtered and standardized stock calcium nitrate and sodium bicarbonate at 25° C. with a calcium and carbonate concentration of the working solution of 2.616×10⁻³ M. The pH of this solution is then adjusted to 8.5 with standardized 0.1M potassium hydroxide solution and this is allowed to equilibrate in temperature and CO₂ partial pressure.

The cleaned scaffold is then suspended in this solution, making sure the solution is fully infused into the scaffold. Ion concentrations are constantly monitored and corrected by the addition of calcium nitrate, sodium carbonate, sodium bicarbonate, and potassium hydroxide, either manually or via a pH stat.

Once the desired level of mineralization is achieved, the matrix is lyophilized to remove residual moisture and to complete the process.

3A Silication of Mycelial Scaffold via Hydrothermal Hot Pressing

When silicate mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur. This prepared scaffold is then deactylated to prepare it for mineralization.

A calcium silicate solution is created from finely ground quartz and calcium oxide, mixed well with a 1:1 Ca:Si ratio and a 20:1 water: powder ratio. The pH of this solution is adjusted to 12 with ammonium hydroxide and is transferred to a nonreactive vessel to be autoclaved at 150° C. for 24 hours to create calcium silicate, likely xonotlite [Ca₆Si₆O₁₇(OH)₂].

The silicate product is then collected, washed with DI water, and dried for 24 hours to remove residual water. The dry silicate powder is then mixed well into a 90 wt % slurry with DI water and is then infused into the prepared scaffold at a slurry: scaffold ratio of 20:1.

The scaffold/slurry matrix is then moderately compressed (upwards of 50 MPa) and returned to the autoclave at 150° C. for upwards of 2 hours. The mineralized product is then dried to complete the process.

3B Silication of Mycelial Scaffold via a Solution-based Reaction

When silicate mineralization is desired upon a bio-scaffold consisting of a cultivated mass of fungal mycelium, one should begin by preparing the mycelial mass for functionalization, taking care to preserve both the cell structure and mycelial matrix, while removing interfering/undesired constituents. This will serve as the raw scaffold, upon which mineralization will occur.

This prepared scaffold is then deactylated to prepare the scaffold for functionalization.

The deacetylation step removes the primary amine from chitin, which cannot be functionalized, and transitions the functional group to a hydroxyl, which is chitosan. This hydroxyl can then serve as the targeted site for phosphorylation or the like.

The deacetylation process uses a 5 molar concentration of NaOH at 90 C for 30 to 120 minutes. The specimen is immersed in solution during this time.

The deactylated scaffold, under air or an inert atmosphere, is then imbibed with catalytic agent (including, but not limited to, an appropriate concentration and type of acid [e.g., acetic acid, hydrochloric acid, phosphoric acid, or the like], fluoride salts (e.g., potassium fluoride, sodium fluoride, tetra-n-butylammonium fluoride, or like like), water, an amino acid (e.g., cysteine or the like) or an amine [e.g., urea, imidazole, or the like]).

The catalyst/scaffold matrix is then infused with tetraethylorthosilicate and silica allowed to condense onto the scaffold for upwards of 24 hours. The product is then dried to complete the process.

The invention thus provides a relatively simple process for producing a mineralized biocompatible material as well as a unique biomedical material that may be used for medical applications in place of mineralized collagen membranes and collagen/hydroxyapatite composite scaffolds. 

What is claimed is:
 1. A structure comprising a scaffold of fungal biopolymer of predetermined form characterized in being formed of a network of interconnected mycelia cells; and a coating of one of hydroxyapatite, calcite and silicate on at least some of said cells within said network.
 2. A structure as set forth in claim 1 wherein said predetermined form is a flat panel shape with a thickness of 2.5 cm.
 3. A process of making a mineralized mycelium scaffolding comprising the steps of obtaining a scaffold of fungal biopolymer having a network of interconnected mycelia cells; and thereafter mineralizing said scaffold.
 4. A process as set forth in claim 3 wherein said fungal biopolymer of said scaffold has a chitin/chitosan backbone and said process further comprises a step of functionalizing said scaffold by removing a —OH moiety therefrom and replacing said moiety with a phosphate group to increase an affinity for cations.
 5. A process as set forth in claim 4 wherein said step of mineralizing said scaffold with at least one of silicates, apatites, and carbonates.
 6. A process as set forth in claim 4 further comprising the steps of placing said functionalized scaffold in a saturated calcium hydroxide solution for a period of time sufficient to create calcium phosphate precursor sites on said mycelia cells of said scaffold; thereafter suspending said scaffold in a solution with ion concentrations 1.5 times that of the human body and a pH of 7.25 for a period of time sufficient to precipitate a hydroxyapatite coating on said cells of said scaffold; and thereafter removing said scaffold from said solution and drying said scaffold.
 7. A process as set forth in claim 4 further comprising the steps of preparing a slurry of milled calcium carbonate and dicalcium phosphate in water; drying said slurry to remove residual water therefrom and to form a desiccated powder; calcinating said powder at temperature and time sufficient to create a hydroxyapatite powder; creating a ceramic slurry with said hydroxyapatite powder, water, a binder, a plasticizer and a dispersant imbibing said functionalized scaffold with said ceramic slurry; lyophilizing said imbibed scaffold to remove moisture therefrom; and thereafter sintering said imbibed scaffold at a temperature and time sufficient to create a ceramic with said predetermined form.
 8. A process as set forth in claim 3 wherein said fungal biopolymer of said scaffold consists of aligned bundles of fungal biopolymer assembled into a predetermined microstructure.
 9. A process as set forth in claim 8 further comprising the steps of functionalizing said scaffold by removing a —OH moiety therefrom and replacing said moiety with a phosphate group to increase an affinity for cations; placing said functionalized scaffold in a saturated calcium hydroxide solution for a period of time sufficient to create calcium phosphate precursor sites on said mycelia cells of said scaffold; thereafter suspending said scaffold in a solution with ion concentrations 1.5 times that of the human body and a pH of 7.25 for a period of time sufficient to precipitate a hydroxyapatite coating on said cells of said scaffold; and thereafter removing said scaffold from said solution and drying said scaffold.
 10. A process as set forth in claim 3 further comprising the steps of preparing a supersaturated solution of calcium nitrate and sodium bicarbonate of a pH of 8.5; suspending said scaffold in said solution for a period of time sufficient to mineralize said scaffold with calcite; and removing said scaffold from said solution and lyophilizing said scaffold to remove residual moisture therefrom.
 11. A process as set forth in claim 3 further comprising the steps of deactyling said scaffold; preparing a calcium silicate solution is of finely ground quartz and calcium oxide with a pH of 12; autoclaving said calcium silicate solution at a temperature and time sufficient to create calcium silicate, drying said calcium silicate to a powder; thereafter mixing said dried silicate powder with water to from a slurry; and infusing said slurry into said deactylated scaffold to form a scaffold/slurry matrix.
 12. A process as set forth in claim 11 wherein said slurry is infused into said deactylated scaffold at a slurry: scaffold ratio of 20:1
 13. A process as set forth in claim 11 further comprising the steps of compressing said scaffold/slurry matrix; heating the compressed scaffold/slurry matrix at 150° C. for up to 2 hours; and then drying the heated compressed scaffold/slurry matrix.
 14. A process as set forth in claim 3 further comprising the steps of deactyling said scaffold; creating a solution of catalytic agent, fluoride salts, water, and one of amino acid and an amine; imbibing said deactylated scaffold with said solution to form a scaffold/slurry matrix; Infusing said matrix with tetraethylorthosilicate for a period of time sufficient to condense said tetraethylorthosilicate onto said matrix; and thereafter drying said matrix. 